Abstract: Fluorescence is the most popular optical contrast for studying live cells. However, fluorescence imaging faces fundamental limitations for probing a vast number of small bio-molecules such as metabolites (e.g., ATP), second messengers, neurotransmitters and drugs. Most of these molecules are intrinsically non-fluorescent. Moreover, labeling them is not feasible, because their biochemical activities would be strongly altered by bulky probes. Thus, how to image these species inside live cells represents a grand challenge. Novel imaging techniques that accomplish this goal would undoubtedly open up new avenues, transforming our ability to monitor biochemistry in living systems in real time. We propose to solve this problem using an emerging multi-photon optical imaging method: stimulated radiation microscopy. By harnessing the power of stimulated Raman scattering (SRS), which serves as a quantum mechanical mechanism for light amplification, chemical contrast from the vibrating chemical bonds in the sample can be generated with high resolution in 3D without adding any external labels. While SRS microscopy is transforming label-free chemical imaging, the technique is still in its infancy. Particularly, both the detection sensitivity and specificity, th key performance criteria, are not high enough for SRS to be truly revolutionary. Many interesting molecules are still beyond detection. We propose to bring the technique to the stage where it can be widely applied to most small bio-molecules. Our plans are: (1) to couple SRS excitation with photo-thermal dark field imaging, a background-free detection scheme estimated to be ~100 times more sensitive; and (2) to use a broadband wavelength multiplex approach to significantly enhance the detection specificity, which should be able to distinguish more closely related chemical species. We are applying stimulated radiation to tackle two compelling problems in lipid biology and neurobiology: (1) Genetic screening for fat-regulating genes by chemical imaging. In order to identify new genes regulating fat metabolism, we will combine SRS lipid imaging with RNA interference screening. We have recently demonstrated such a novel combination of imaging and genetics with C. elegans. The proposed sensitivity boost would expand the screening to the genome scale, and the specificity enhancement should allow us to probe unsaturated lipid and cholesterol. (2) Optical monitoring of membrane potentials. Despite of many efforts, there is no satisfactory optical method to monitor voltage signal in neurons. The intense electric field across the plasma membranes during action potentials should shift the vibrational frequency of membrane lipids, and we plan to employ this vibrational electrochromism as a label-free contrast mechanism for voltage imaging. The proposed technical innovation has the potential to greatly advance light microscopy, lipid biology, genetic screening and neuroscience, and the applications will take bio-imaging into new areas of biomedicine that have been previously uncharted. Public Health Relevance: The unprecedented ability to visualize small molecules such as metabolites and drugs in living cells and organisms without any labels will revolutionize many areas of biomedical research, particularly lipid biology, pharmacokinetics and cancer diagnosis. The proposed genetic screening research would discover genes that regulate fat metabolism and distribution on the multicellular organism models. These newly identified genes could become potential drug targets for combating obesity and related metabolic disorders.